Abstract
The family of Notch proteins plays a key role in cell fate determination. Additionally, Notch proteins regulate critical functions of the endothelium, as well as other recruited supporting cells, in concert with other pathways. Despite significant advances in the field and extensive studies focused on elucidating this pathway, many questions remain regarding Notch activation and its upstream/downstream effects, with vascular biology constituting one area of particular interest. Here, we provide a brief description of the components and functions of the Notch pathway in vasculature, followed by a detailed compilation of recommended methods of evaluation in vitro and in vivo. We provide a rationale for key elements when choosing different approaches and controls, strengths and limitations, and essential considerations when providing a meaningful interpretation of results. Our aim is to describe a careful approach to assessing Notch function in endothelial cells, based on underlying principles, with the overall goal of obtaining physiologically relevant information that will enhance our understanding of this pathway and its role in vascular biology.
Highlights
The Notch pathway plays a number of essential roles within the field of vascular biology: it is critical for arterial differentiation, maturation, and quiescence, as well as cardiovascular function, among other processes
We found that luciferase expression peaks between 6 to 8 hours after 5mM Ethylenediaminetetraacetic acid (EDTA) stimulation in HUVECs compared to Hank’s buffered saline solution (HBSS) controls (9.4±1.3 vs 74.1±26.5 Relative Luciferase/ Renilla units, p
To give an example of how these mouse models could be used: we previously studied whether vascular endothelial growth factor A (VEGF-A) inhibition leads to Notch activation within the tumor vasculature of neuroblastoma xenografts [104]
Summary
The Notch pathway plays a number of essential roles within the field of vascular biology: it is critical for arterial differentiation, maturation, and quiescence, as well as cardiovascular function, among other processes. A cleaved Notch (cN1) antibody (Cell Signaling Technology, Danvers, MA, USA) that only detects the residue exposed after γ-secretase cleavage at S3, Val1744 in humans and mice, and runs at 110 kDa in SDS-PAGE is an excellent way to quantify activated Notch, when normalized to a housekeeping control such as β-actin. In EDTA stimulated HUVECs incubated for either four or six-hours, we found RNA-seq revealed differentially expressed genes (DEGs) which included canonical downstream targets such as Hes (2.80-fold ± 0.07, p
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